The UK company beat Next, Very, The Outnet and ASOS to take home the prize from Lorraine Kelly's ITV breakfast show.
Boohoo joint-chief executive Carol Kane said: "We are delighted to have been recognised as the best online retailer by the viewers of Lorraine, especially within such a strong category.
"Winning any award is always an honour, but there is something particularly special about winning when the public have voted."
Boohoo was praised for its unique supply chain, which means that clothing, accessories and shoes go from drawing boards to customers in a matter of weeks.
Since launching in 2006, it has become one of the fastest-growing online clothing businesses in the UK and Europe.
Other winners at the High Street Fashion Awards included Primark and French Connection, who landed the 'Best Affordable Fashion' and 'Best Premium Brand' gongs respectively, and Topshop, which was named 'Best Trendsetter'.
Kelly commented: "The High Street Fashion Awards are always great because we get to see what brands the viewers are really passionate about. We should all be championing our local high street and the awards are an excellent way to show our support."
Louise Redknapp, who judged the awards alongside Lorraine stylist Mark Heyes and Look editor Ali Hall, added: "The high street works for me, I'm a real last-minute shopper and I know you can pretty much go into any shop on the high street and find what you're looking for. We are so well set up here in the UK and the high street is great."